The present invention relates generally to compositions and methods for treating immune system mediated diseases such as rheumatoid arthritis (RA). RA is a destructive autoimmune disease that is a chronic inflammatory and destructive arthropathy of unknown etiology. RA is commonly associated with decreased life expectancy. RA causes the synovial lining to become hyperplastic leading to formation of pannus and destruction of cartilage and bone. In fact, RA may cause the synovial lining to increase from 1-2 cell layers to as much as 10 layers thick. The increase in synovial lining may be attributed to decreased death of both fibroblasts and macrophages since there is a paucity of apoptotic cells in RA, even though the milieu of the joint contains noxious factors that are normally detrimental to the survival of the cell. The induction of synoviocyte apoptosis in animal models of inflammatory arthritis, including streptococcal cell wall-induced arthritis, collagen-induced arthritis, HTLV-1 tax transgenic model, and RA explants in SCID mice result in either amelioration of the disease or reduction in joint inflammation and destruction. While the data from these animal studies suggest that increased apoptosis may be associated with an improved clinical outcome, the studies use adenoviral vectors, overexpression of FasL, or anti-Fas antibodies that are known to induce an inflammatory response. Thus, the applicability of these studies as a basis for therapies to treat RA is unclear and questionable.
Apoptosis in mammals proceeds through two distinct pathways, an “extrinsic” pathway that transduces an apoptotic signal following the ligation of death receptors on the cell surface and an “intrinsic” pathway in which mitochondria play a critical role. The induction of apoptosis mediated by the extrinsic pathway is initiated by binding of death ligands to their receptors. The intrinsic pathway is regulated by the Bcl-2 protein family which is divided into anti-apoptotic members (Bcl-2, Bcl-xL, Mcl-1, Al/Bfl-l and Bcl-w) and pro-apoptotic members (Bax, Bak, Bad, Bim/Bod, Bok/Mtd, Bik/Blk/Nbk, Bid, Hrk/DP5, Bmf, Noxa, Puma/Bbc3). Bcl-2-related proteins contain Bcl-2-homology (BH 1-4) domains that are critical for homodimer and heterodimer formation between the family members. While the anti-apoptotic Bcl-2 like proteins contain at least three and possibly all four BH domains, the pro-apoptotic Bcl-2 related proteins are subdivided into two categories: (1) the multi-BH domain (BH1-3: e.g. Bak, Bax); and (2) the BH3-only proteins (e.g. Bad, Bim).
Many Bcl-2 family members are localized to the mitochondrial outer membrane and certain other intra-cellular membranes which suggest that mitochondrial dysfunction is involved in apoptosis. During intrinsic apoptosis signaling, the integrity of the outer mitochondrial membrane is lost, leading to the dissipation of the transmembrane potential through the opening of mitochondrial permeability transition pores and release of apoptogenic mitochondrial inter-membrane proteins, such as cytochrome c. In the cytoplasm, cytochrome c binds to the adaptor protein Apaf-1 which then causes aggregation and activation the initiator caspase 9. Caspase 9, in turn, activates the effector caspases 3 and 7 that cause the downstream degradative events in apoptosis. Apoptosis signaling through the intrinsic pathway is inhibited by overexpression of any of the Bcl-2 like pro-survival members or by loss of both multi-BH domain proteins Bak and Bax.
The BH3 domain is critical for cell death since deletion of the BH3 domain results in a failure to induce apoptosis in cells overexpressing the mutant constructs. The BH3 domain forms an amphipathic α-helix that binds to hydrophobic cleft on the surface formed by the BH1-3 domain of the anti-apoptotic Bcl-2 family members. Recent studies using peptides that correspond to the BH3 domains have shown that BH3-only proteins are also subdivided into two categories based on their ability to induce apoptosis. Bid and Bim are sufficient to sequester anti-apoptotic Bcl-2 family members, induce oligomerization of Bak and Bax, induce permeabilization of liposoines, and/or the release of cytochrome C. In contrast, Bad, Bmf, Hrk, Noxa, and Puma are sensitizers for apoptosis since they are only able to bind to the anti-apoptotic Bcl-2 members and require Bid or Bim to induce the death response.
Recent studies have examined the potential of altering the molecular rheostat that governs the Bcl-2 family through the use of BH3-domain peptides. However, to date, all studies that used BH3-peptidometrics have only examined their efficacy in xenograph tumor models and in immune-incompetent mice. Furthermore, since an increasing number of patients have failed to respond to traditional biologic therapy, which has a mode of action associated with increased apoptosis in the joint, it is clear that additional therapies are warranted.
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